Accumulating evidence indicates that the ATP-coupled covalent ligation of the low molecular weight polypeptide, ubiquitin, to various target proteins within eukaryotes is of fundamental importance to cellular regulation. The long-term objective of the proposed research is to understand the function of this novel post- translational modification in cell regulation, with emphasis on its role in energy-dependent protein degradation. The studies will test a model for the role of ubiquitin in protein turnover which proposes that the observed specificity of degradation of short- lived and abnormal proteins results from the partitioning of the conjugate pool between alternate fates of degradation and disassembly, in which the ubiquitin moiety is non-productively cleaved. The basis for this partitioning is hypothesized to depend on the degree of target protein structural instability induced by the convalent conjugation of ubiquitin; therefore, the degradative specificity would principally reside in features of the target protein and not in the step of ubiquitin ligation. The model predicts that both short-lived and long-lived proteins should be found conjugated, but that the two pools of ubiquitin-ligated target proteins should partition differently between degradation and disassembly. In addition, the model predicts that ubiquitination should lead to a generally enhanced proteolytic susceptibility of the target protein. Two independent, complimentary approaches will be taken. Immunochemical methods will be used to quantitate and isolate pulse-labeled free and conjugated ubiquitin pools to determine the range of protein half lives for target proteins within cultured human lung fibroblasts. Because of the significant rate of ubiquitin turnover, these methods will be used to correct for label incorporation into the ubiquitin moiety of the isolated conjugates. Other studies will identify and characterize the mechanism of action for a serum factor shown to induce net increases in both free and conjugated ubiquitin pools within the fibroblasts. The enzymes catalyzing ubiquitin conjugation will be purified from rabbit liver then characterized in terms of their function and enzymatic mechanism. These pure conjugating enzymes will then be used for in vitro model studies to test whether ubiquitination of target proteins induces a general enhanced proteolytic susceptibility as its mechanism of action.